Novel immunoassay instrumentation based on the electrochemical detection of enzymatically-generated species is proposed. The system will incorporate highly selective chemically modified electrodes as amperometric sensors to improve detection limits and reduce the probability of interferences. The modified electrodes act as catalysts to lower the overpotential required for detection, but their use in biomedical analysis has not been significantly explored. In Phase I or the SBIR program, several types of modified surfaces will be synthesized. We will investigate adsorption of redox polymers (e.g. polydopamine), reductive transition metal complex polymerization, and monomer reduction/ligand exchange as three possible means to attach suitable redox mediators to the electrode surface. Each scheme will be evaluated by cyclic voltammetry, rotating disc electrode voltammetry and chronocoulometry to assess half-wave potentials, surface coverage of the mediator, and electrocatalysis. The electrodes will be tested as sensors on a flowing stream for electrode stability and their suitability to detect NADH catalytically. A simple prototype flow injection analyzer incorporating the most likely electrodes will be constructed in the last half of Phase I and enzyme-multiplied immunoassays of blood plasma containing either phenytoin or digoxin will be carried out using commercial kits and this electrochemical system as well as conventional ultraviolet detection. It is anticipated that the proposed system will allow sample throughput of at least 60 per hour, require much shorter incubation times for NADH production than either UV or fluorescence detection, and attain detection limits for NADH of less than 100 femtomoles injected. Using modified electrodes as the transducer will further improve selectivity and attenuate noise by reducing the overpotential required for detection. The new analyzer would extend the utility of non-radiolabelled immunoassay to the sub-nanogram/mL concentration range without sacrificing reliability or extending the enzyme-amplification incubation time past a few minutes. Since electrochemical detection is roughly three orders of magnitude more sensitive than ultraviolet absorbance, even low picogram/mL levels of circulating hormones should theoretically be detectable if longer incubation times are used.